The present invention relates to the use of an antagonist of OSM in the manufacture of a medicament for the treatment or prophylaxis of an inflammatory arthropathy or inflammatory disorder and methods of screening for such antagonists.
Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects articular joints, characterised by synovial hyperplasia and extensive cellular infiltration by mononuclear cells and polymorphonuclear leukocytes(PMN). A complex, and poorly understood interplay between resident and infiltrating cell types leads to the chronic secretion of metalloproteinases(MMPs), resulting in destruction of articular cartilage, ligaments and subchondral bone (Firestein G S Current Opinion in Rheumatology. 4:348-54, 1992). Among the numerous pro-inflammatory cytokines implicated in driving RA joint pathology, TNFxcex1 has been shown to play a pivotal role, with anti-TNFxcex1 therapies showing clear benefit (Elliott M J. et al. Lancet. 344(8930):1105-10, 1994). TNFxcex1 mediates several pathologic effects including induction of MMPs (Dayer J M. et al Journal of Experimental Medicine. 162(6):2163-8, 1985), upregulation of other pro-inflammatory cytokines (Haworth C. et al. European Journal of Immunology. 21(10):2575-9, 1991 and Dinarello C A. et al. Journal of Experimental Medicine.163(6):1433-50, 1986) and increased PMN adhesion and transendothelial cell migration(Smart S J. Casale T B American Journal of Physiology. 266:L238-45, 1994). Though TNFxcex1 is viewed currently as the initiator of the pro-inflammatory cytokine cascade, relatively little is known of its positive regulation (Feldmann M. et al. Annual Review of Immunology. 14:397-440, 1996).
Oncostatin M (OSM) (Rose T M. Bruce A G. PNAS USA 88(19):8641-5, 1991) is a 28 kDa glycoprotein which belongs to a family of cytokines comprising IL-6, IL-11, leukaemia inhibitory factor (LIF), cililiary neurotrophic factor (CNTF) and cardiotrophin 1 (CT-1)(Taga T. Kishimoto T. Annual Review of Immunology. 15:797-819, 1997). All members share a common signalling chain, gp130, as part of a complex family of hetero- and homodimeric receptors (Grotzinger J. et al. [Article] Proteins. 27(1):96-109, 1997). OSM shares a common heterodimeric receptor with LIF, (LIFr: gp130, type I) and also has its own unique receptor comprising OSMrxcex2 chain and gp130 (type II) (Mosley B. et al. [Article] Journal Of Biological Chemistry. 271(51):32635-32643, 1996). OSM has long been known for effects on cell growth and differentiation (Horn D. et al [Journal Article]Growth Factors. 2(2-3):157-65, 1990). Recently, OSM has also been shown to have potent, pro-inflammatory properties in mice in vivo (Modur V. et al. J. Clin Invest. 100:158-168, 1997) and demonstrates potent synergy with IL-1 to promote articular cartilage degradation in model systems, ex-vivo (Cawston T. Biochemical and Biophysical Research Communications. 215(1):377-85, 1995).
OSM induces a prolonged increase in P-selectin (and E-selectin) in endothelial cells (Yao L. et al. Journal Of Experimental Medicine. 184(1):81-92, 1996), stimulates urokinase-type plasminogen activator activity in human synovial fibroblasts (Hamilton J. et al Biochemical and Biophysical Research Communications. 180(2):652-9, 1991) and is a powerful inducer of IL-6 from endothelial cells(Brown Tj.et al. Journal Of Immunology. 147(7):2175-80, Oct. 1, 1991). OSM has recently been measured in RA but not OA synovial fluid (Hui W. et al. Annals Of The Rheumatic Diseases. 56(3):184-187, 1997) and synovium, production of which has been localised to macrophages (1 997, Okamoto H et al. Arthritis and Rheumatism 40(6): 1096-1105) and Cawston et al (1998, Arthritis and Rheumatism, 41(10) 1760-1771). To-date further experiments in this field have been speculative based on the similarity of the IL-6 subfamily members (Carroll G. et al Inflamm. Res. 47 (1998) 1-7).
The present inventors have discovered that OSM has the ability to induce TNFxcex1 secretion in macrophages. Contrary to recent data suggesting that OSM upregulates production of tissue inhibitor of metalloproteinase-1 (TIMP-1) (Nemoto et al 1996, AandR 39(4), 560-566), which complexes with and inactivates MMP-1 and would therefore be expected to decrease collagen release, the inventors discovery that OSM induces TNFxcex1 secretion suggested to them that OSM may actually play a role in mediating cartilage destruction. Based on this discovery, the present inventors have demonstrated that therapeutic administration of a neutralising anti-OSM antibody without inhibition of other IL-6 family members can alone ameliorate collagen-induced arthritis in a mouse model. Synergy of OSM with TNFxcex1 to promote collagen release from cartilage has subsequently been shown by T. Cawston et al (1998, Arthritis and Rheumatism, 41(10) 1760-1771).
According to the present invention there is therefore provided the use of an antagonist of OSM in the manufacture of a medicament for the treatment or prophylaxis of an inflammatory arthropathy or inflammatory disorder. A particular use of an antagonist of OSM is in the manufacture of a medicament to prevent or reduce collagen release from cartilage. The invention further provides a method for the treatment or prophylaxis of an inflammatory arthropathy or inflammatory disorder comprising administering an effective amount of an antagonist of OSM to a patient suffering from such a disorder.
The antagonist may function by blocking OSM from interaction with the OSM receptor gp130, or the other OSM receptors, OSMrxcex2 chain or LiFr, or by blocking formation of heterodimers of these proteins, and as such prevent OSM binding and signalling thereby reducing synthesis of pro-inflammatory cytokines and/or MMPs. The antagonist according to the invention may therefore be a ligand for either OSM or one or more of the OSM receptors (gp130, OSMrxcex2 or LIFr) or an agent capable of interfering with these interactions in a manner which affects OSM biological activity. Hereinafter reference to an antagonist to OSM can be taken to mean either an antagonist to OSM itself or to one of its receptors.
The present inventors have also demonstrated that in rheumatoid artritis synovial vascular endothelium, P and E-selectin co-localise with gp130, the sionalling element of type I and II OSM receptors. Without wishing to be bound by theory this indicates that OSM, produced by synovial macrophages might prime RA vascular endothelium to facilitatc leucocyte recruitment via upregulation of P and E-selectin. The finding that ligation of L-selectin by either specific antibody or fucoidan (L-selectin agonist) drives human mononuclear cells to secrete OSM may be highly significant in terms of amplification of the inflammatory response, by providing an additional local source of OSM to drive TNFxcex1 and P and E-selectin.
Amino acid residues which are important for OSM""s interaction with gp130 have been identified. From the published amino acid sequence of OSM (Malik et al., 1989, Mol. Cell Biol., 9(7), 2847-53, DNA sequence entry M27288 in EMBL database, protein sequence entry P13725 in Swissprot) these are G120, Q16 and Q20; N123 and N124 may also play a part (see SEQ ID. 12 and below). The first 25 residues are a signal peptide, and the mature protein begins at the sequence AAIGS. (SEQ ID NO: 13). The sequence is numbered from the first amino acid of the mature protein as shown.
The invention therefore further provides an antagonist or agent capable of interacting with one or more of these specific residues and or the binding sites they help to define on OSM to alter OSM biological activity.
Inflammatory arthropathies which may be treated according to this invention include rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, inflammatory osteoarthritis and/or reactive arthritis. Inflammatory disorders which may be treated include, amongst others, Crohns disease, ulccerative colitis, gastritis for example gastritis resulting from H. pylori infection, asthma, chronic obstructive pulmonary disease, alzheimer""s disease, multiple sclerosis and psoriasis.
Potential antagonists of OSM include small organic molecules, ions which interact specifically with OSM for example a substrate possibly a natural substrate, a cell membrane component, a receptor or a natural ligand, a fragment thereof or a peptide or other proteinaceous molecule, particularly preferred is a non-signalling mutant form of OSM which will block binding of OSM to the OSM receptor, but also modified OSM molecules. Such antagonists may be in the form of DNA encoding the protein or peptide and may be delivered for in vivo expression of said antagonists. Antagonists may be vaccines comprising such protein or peptide molecules or DNA, designed to produce an antagonistic effect towards OSM via induction of antibody responses in vivo targeted towards native OSM. Such antagonists may also include antibodies, antibody-derived reagents or chimaeric molecules. Included in the definition of antagonist is a structural or functional mimetic of any such molecule described above. Also contemplated are nucleic acid molecules such as DNA or RNA aptamers.
Preferred antagonists include small organic molecules. Such compounds may be from any class of compound but will be selected on the basis of their ability to affect the biological activity of OSM through one of the mechanisms described above and will be physiologically acceptable ie non-toxic or demonstrating an acceptable level of toxicity or other side-effects. One class of compounds which may provide useful antagonists are ribonucleosides such as N-(1H-pyrazole[3,4-d]pyrimidin-4yl)benzamide); Davoll and Kerridge, J. Chem Soc., 2589, 1961)
Other preferred antagonists include antibodies, fragments thereof or artificial constructs comprising antibodies or fragments thereof or artificial constructs designed to mimic the binding of antibodies or fragments thereof. Such constructs are discussed by Dougall et al in Tibtech 12, 372-379) (1994).
Also included in the definition of antibody are recombinant antibodies such as recombinant human antibodies, which may be used. The antibodies may be altered ie they may be xe2x80x9cchimaericxe2x80x9d antibodies comprising the variable domains of a donor antibody and the constant domains of a human antibody (as described in WO86/01533) or they may be xe2x80x9chumanisedxe2x80x9d antibodies in which only the CDRs are derived from a different species than the framework of the antibody""s variable domains (as disclosed in EP-A-0239400). The complementarity determining regions (CDRs) may be derived from a rodent or primate monoclonal antibody. The framework of the variable domains, and the constant domains, of the altered antibody are usually derived from a human antibody. Such a humanised antibody should not elicit as great an immune response when administered to a human compared to the immune response mounted by a human against a wholly foreign antibody such as one derived from a rodent.
Preferred antagonists include complete antibodies, F(abxe2x80x2)2 fragments, Fab fragments, Fv fragments, ScFv fragments, other fragments, CDR peptides and mimetics. These can be obtained/prepared by those skilled in the art. For example, enzyme digestion can be used to obtain F(abxe2x80x2)2 and Fab fragments (by subjecting an IgG molecule to pepsin or papain cleavage respectively). References to xe2x80x9cantibodiesxe2x80x9d in the following description should be taken to include all of the possibilities mentioned above.
As will be appreciated by those skilled in the art, where specific protein or peptide antagonists are described herein, derivatives of such antagonists can also be used. The term xe2x80x9cderivativexe2x80x9d includes variants of the antagonists described, having one or more amino acid substitutions, deletions or insertions relative to said antagonists, whilst still having the binding activity described. Preferably these derivatives have substantial amino acid sequence identity with the antagonists specified.
The degree of amino acid sequence identity can be calculated using a program such as xe2x80x9cbestfitxe2x80x9d (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) to find the best segment of similarity between any two sequences. The alignment is based on maximising the score achieved using a matrix of amino acid similarities, such as that described by Schwarz and Dayhof(1979) Atlas of Protein Sequence and Structure, Dayhof, M. O., Ed pp 353-358.
Preferably the degree of sequence identity is at least 50% and more preferably it is at least 75%. Sequence identities of at least 90% or of at least 95% are most preferred. It will nevertheless be appreciated by the skilled person that high degrees of sequence identity are not necessarily required since various amino acids may often be substituted for other amino acids which have similar properties without substantially altering or adversely affecting certain properties of a protein. These are sometimes referred to as xe2x80x9cconservativexe2x80x9d amino acid changes. Thus the amino acids glycine, valine, leucine or isoleucine can often be substituted for one another include:xe2x80x94phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); asparate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains) and cysteine and methionine (amino acids having sulphur containing side chains). Thus the term xe2x80x9cderivativexe2x80x9d can also include a variant of an amino acid sequence comprising one or more such xe2x80x9cconservativexe2x80x9d changes relative to said sequence.
The present invention also includes fragments of the antagonists of the present invention or of derivatives thereof which still have the binding activity described. Preferred fragments are at least ten amino acids long, but they may be longer (e.g. up to 50 or up to 100 amino acids long).
Further preferred antagonists of OSM for use in the invention are oligonucleotide ligands. Systematic evolution of ligands by exponential enrichment (SELEX) is a protocol in which vast libraries of single stranded oligonucleotides are screened for desired activity against a target protein or other molecule (Tuerk and Gold 1990 Science 249, 505-510, Green et al, 1991 Meths. Enzymol. 2 75-86; Gold et al., 1995 Annu. Rev Biochem 64,763-797; Uphof et al., 1996 Curr. Opin. Struct. Biol. 6, 281-288). The product of this screen is a single oligonucleotide sequence termed an aptamer with desired activity, usually high affinity binding, for the target protein. The SELEX procedure is usually initiated with an RNA or DNA library consisting of some 1014-1015 random oligonucleotide sequences. In a fully randomised oligonucleotide library, each molecule will exhibit a unique tertiary structure which will be entirely dependent on the nucleotide sequence of that molecule. Thus when screened against a target protein the binding affinity of the oligonucleotide for that protein will be determined by the fit between the shape of the oligonucleotide and epitopes on the target protein. As a consequence of starting from a library of vast diversity it is usual to be able to identify aptamers of sub-nM affinity for the target protein with selectivity for that target protein over other proteins with overall structural homology (Tuerk and Gold 1990 supra, Green et al, 1991 supra; Gold et al., 1995 supra; Uphof et al., 1996 supra). Using SELEX methodology RNA or DNA aptamers have been generated to over 100 proteins and small molecules including dopamine (Mannironi et al, 1997 Biochemistry 36,9726-9734), substance P (Nieuwlandt et al, 1995 biochemisry 34, 5651-5659), human neutrophil elastase (Bless et al., 1997 Current biol. 7, 877-880), Platelet Derived Growth Factor (PDGF) (Green et al, 1996 Biochemisry 35, 14413-14424), Vascular Endothelial Growth Factor (VEGF) (Green et al., 1995 Chem Biol. 2, 683-695), thrombin (Bock et al., 1992 Nature 355, 564-66) and L-selectin (O""Connell et al., 1996 PNAS USA 93,5883-5887).
The invention therefore provides aptamers capable of binding to OSM (or an OSM receptor) in particular RNA aptamers. Preferred are the aptamers listed in FIG. 16 more particularly Family C aptamers and most particularly aptamer ADRS58 as described in Example 11a.
A number of aptamers have been demonstrated to have biological activity, usually receptor antagonism or enzyme inhibition, both in vitro and in vivo. For example RNA aptamers with high affinity and inhibitory activity to human neutrophil elastase (hNE) were generated by blended SELEX (Bless et al., 1997 supra ). Following post-SELEX modification to increase in vivo stability the aptamer was tested in a rat model of lung inflammation (Bless et al., 1997supra). In a second example, a 49 nucleotide long DNA aptamer was generated to human L-selectin with nM affinity for the protein (O""Connell et al., 1996 supra). The aptamer exhibits 600-fold selectivity for L-selectin over E-selectin and 10000-fold selectivity over P-selectin. Intravenous injection of a PEG formulation of the aptamer inhibited trafficking of radiolabelled human PBMC to the lymph nodes, but not to other organs, in a dose dependent manner (Hicke et al., 1996 J. Clin. Invest. 98,2688-2692). In a third example high affinity RNA aptamers have been raised against human VEGF to investigate the role of VEGF in angiogenesis (Jellinek et al., 1994 Biochemistry 33, 10450-10456; Green et al., 1995; Ruckman et al., 1998. J. Biol. Chem 273,20556-20567; Willis et al., 1998 Bioconjug. Chem. 9,573-582). A liposomal formulation of the VEGF aptamer inhibits VEGF induced endothelial cell proliferation in vitro and vescular permeability increase and angiogenesis in vivo (Willis et al., 1998 supra). There is therefore provided an oligonucteotide ligand in particular an aptamer especially an RNA aptamer as descirbed above of OSM or an OSM receptor (OSMR, LIFR, gp130) for use in the invention.
To raise an aptamer for use in the invention as described above OSM or a receptor must first be bound to plates for screening. Iterative rounds of selection and amplification (ie the SELEX procedure) can then be performed in accordance with Fitzwater and Polisky (Meths in Enzymol. 267, 275-301) to generate RNA or DNA aptamers to human OSM. Typically these aptamers are modified RNA aptamers as RNA provides the greatest structural diversity and therefore possibility of generating high affinity molecules. Following the generation of a high affinity aptamer a number of post-SELEX optimisation protocols may be performed to increase aptamer stability, to truncate the aptamer to a core sequence (typically aptamers are 100 mers or shorter) that is more amenable to solid phase synthesis thereby reducing the cost of synthesis, and to develop formulations for use in vivo.
In the first of these procedures the aptamer may be truncated to reduce the length of the molecule to a core sequence required for activity. The short core sequence, often between 20 and 40 nucleotides long, will be cheaper and quicker to synthesise and may have increased bioavailability. Information regarding the composition of the core sequence may be obtained from sequence homology comparisons. However, truncation experiments usually involve the synthesis of sequentially shorter aptamers until a minimum sequence required for activity is generated. This usually involves removal of the fixed sequences but there are numerous examples where nucleotides within the fixed sequence have contributed to aptamer affinity (Fitzwater and Polisky, 1996 supra; Ruckman J, et al (1998) J. Biol. Chem. 273, 20556-20567. Green et al., 1995 supra). The invention may therefore provides aptamers which are truncated or extended versions of the selected aptamer or one demonstrating greater than 70% homology in sequence to a selected aptamer.
Following truncation a number of base modification experiments may be performed to improve aptamer stability by protection against ribonuclease cleavage. During SELEX it is not possible to include 2xe2x80x2 modified purine bases as the T7 polymerase used for in vitro transcription will not tolerate this modification. Hence to increase aptamer stability post-SELEX it is usual to replace the purine bases within the aptamer with 2xe2x80x2 modified purines. This modification is usually through the use of 2xe2x80x2-0-methyl purines although other modified purines including 2xe2x80x2-amino purines or 2xe2x80x2-fluoro purines may be used (Ruckman et al., 1998 supra; Green et al., 1995 supra). This has to be done in a sequential manner as this modification, post-SELEX, may also result in a loss of affinity (Green et al., 1995 supra).
Following truncation and stabilisation it is possible to generate very large amounts of a short fully modified aptamer that may be synthesised by chemical solid scale synthesis. Many molecules can be added to the 5xe2x80x2 end of an aptamer to facilitate aptamer use or to formulate an aptamer for in-vivo delivery. This includes a caged moiety to aid imaging (Hnatowich D. J. (1996) Q. J. Nucl. Med. 40, 202-8.), fluorescein to aid molecular detection (German et al., 1998 Anal. Chem. 70, 4540-5.), a lipid group to aid insertion into a liposome (Willis et al., 1998 supra), or conjugation to a small molecule drug or peptide (Charlton J, et al (1997b) Biochemistry 36, 3018-3026). Generally, the addition of a molecule to the 5xe2x80x2 end of an aptamer does not result in a loss of affinity or specificity.
To improve in vivo half-life, aptamers have been modified through the addition of polyethylene glycol (PEG) molecules or through the incorporation into liposomes. In both cases such modification can cause a significant increase in in vivo half-life (Willis et al, 1998 supra).
In addition to liposomal formulations aptamers have been formulated with both 20K and 40K PEG to increase serum stability in vivo. A DNA aptamer has been generated against human L-selectin. To increase in vivo stability a 20K PEG ester was coupled to the aptamer through the N-terminal amine moiety. The PEG conjugated aptamer was demonstrated to block L-selectin-dependent lymphocyte trafficking in vivo in SCID mice (Hicke et al., 1996 supra). There is therefore provided for use in the invention, a conjugate of an aptamer and a carrier molecule for example PEG. In this embodiment the aptamer and carrier will be linked for example through the N-terminal amine moiety. In addition there is provided a formulation or composition for use in the invention comprising an aptamer and a delivery molecule for example a liposome. In this embodiment there may be no link between the aptamer and the carrier, the aptamer may simply be encapsulated, dispersed or distributed through the carrier.
The aptamers isolated in this study may also be modified for use as diagnostic molecules to detect the presence of human OSM in serum, tissue or other ex vivo samples, or for the detection of human OSM in whole body in vivo imaging studies (Charlton J,et al (1997) Chemistry and Biology 4, 809-816.; Hnatowich, 1996 supra). Fluorescein or other fluorescent detection groups can be added to the 5xe2x80x2 end of the aptamer molecule to aid in fluorescence detection for applications such as FACS (Fluorescence Activated Cell Sorting) (Davis K A. Et al (1996) Nuc. Acids Res. 24, 702-6.; Charlton et al., 1997supra), ELONA (Enzyme Linked Oligonucleotide Assays) assays (Drolet D W, et al (1996) Nature Biotech. 14, 1021-1025) and other diagnostic applications. The advent of technetium-99m (Tc99 m) chelating peptide cages, such as the MAG3 (Fritzberg A. R.,et al J Nucl Med 1986: 27, 111-6) has greatly facilitated the use of a wide range of molecules (Kubo A.et al, (1998) Kaku Igaku 35, 909-28) and macromolecules (Taillefer R.et al, (1995) Eur. J. Nucl. Med. 22, 453-64.), for imaging the presence of the target protein in vivo (macromolecules (Pallela V. R., et al (1999) Nucl. Med. 40, 352-60.). Images are visualised with the aid of a xcex3-camera and have been achieved in a variety of species from mouse to man. Recent modification of the Tc99m chelators has enabled more efficient and stable labeling of molecules under mild conditions (Hnatowich D. J.1998 Nucl Med 39, 56-64.). Methods for radiolabeling single-stranded oligonucleotides have already been developed, the fate of such unmodified labeled oligonucleotides in vivo has been preliminary investigated (Hnatowich, 1996 supra).
It will of course be appreciated that any peptide, protein or nucleic acid based antagonists for use in this invention will preferably be in a purified form ie free from matter associated with such a molecule either in its natural state or as a result of its manufacture, notably the purity is greater than 70% pure but more preferably greater than 80% or 90% pure.
The antagonists of the present invention may be used alone or in combination with immunosuppressive agents such as steroids (prednisone etc), cyclophosphamide), cyclosporin A or a purine analogue (e.g. methotrexate, 6-mercaptopurine, or the like), or antibodies such as an anti-lymphocyte antibody or more preferably with a tolerance-inducing, anti-autoimmune or anti-inflammatory agent such as a CD4+T cell inhibiting agent e.g. an anti-CD4 antibody (preferably a blocking or non-depleting antibody), an anti-CD8 antibody, an anti-CD23 antibody, a TNF antagonist e.g. an anti-TNF antibody or TNF inhibitor e.g. soluble TNF receptor, or agents such as NSAIDs or other cytokine inhibitors.
Suitable dosages of an antagonist of the present invention will vary, depending upon factors such as the disease or disorder to be treated, the route of administration and the age and weight of the individual to be treated and the nature of the antagonist. Without being bound by any particular dosages, it is believed that for instance for parenteral administration, a daily dosage of from 0.01 to 20 mg/kg of an antibody (or other large molecule) of the present invention (usually present as part of a pharmaceutical composition as indicated above) may be suitable for treating a typical adult. More suitably the dose might be 0.1 to 5 mg/kg, such as 0.1 to 2 mg/kg. A unit dose suitably will be 1-400 mg. Suitable dosages of small organic molecules would be similar and suitable dosages of oligonucleotide ligands would be for example 0.1-10 mg/kg.
The invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an antagonist according to the invention and optionally another therapeutic agent as described above. The antagonist, and pharmaceutical compositions thereof of this invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly or intravenously but depending on the nature of the antagonist other routes such as oral, by inhalation, intra-nasal, topical, or intra articular may be more appropriate.
The compositions for parenteral administration will commonly comprise a solution of the antagonist or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These compositions may be sterilised by conventional, well known sterilisation techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjustment agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody or other antagonist in these formulations can vary widely, for example from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
Thus, a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water, and 50 mg of antagonist. A typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer""s solution, and 150 mg of antibody or other antagonist according to the invention. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington""s Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980). Suitable formulations for nucleic acid antagonists are discussed above.
The protein antagonists of this invention such as antibodies can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins. Any suitable lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of antibody activity loss (e.g., with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted to compensate.
Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of the antibody or other antagonist of this invention sufficient to effectively treat the patient.
The present invention includes within its scope an assay for determining whether or not a particular agent which binds to OSM may be useful in the treatment of an inflammatory disease. The invention therefore comprises an assay for the identification of antagonists of OSM comprising combining OSM with the test agent and determining whether or not the agent is capable of blocking the interaction between OSM and the OSM receptor or affecting OSM biological activity through differential expression of a marker molecule.
To select an antagonist for use in the invention as described above OSM, the key binding residues of OSM as described above presented on a carrier or in a manner in which the binding sites are defined (xe2x80x9cOSM binding moietyxe2x80x9d), or an OSM receptor must first be obtained. cDNA encoding human OSM may be generated synthetically, based on the EMBL sequence (accession number M27288), cloned into an appropriate expression vehicle and used to transform an appropriate host such as E. Coli. Human OSM protein is then purified from culture medium and bound to plates for screening.
OSM, an OSM binding moiety and/or an OSM receptor may be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. The invention therefore provides an assay for the identification of an antagonist of OSM which comprises contacting OSM with a test agent and measuring for binding. These substrates and ligands may be natural substrates and ligands may be structural or functional mimetics. Such molecules are included in the definition of antagonists of OSM. The method of screening may involve high-throughput. For example, to screen for antagonists, a synthetic reaction mix, cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, may be prepared from a cell that expresses OSM receptor. The preparation is then incubated with labelled OSM in the absence or the presence of a candidate molecule. The ability of the candidate molecule to bind to OSM receptor is reflected in decreased binding of the labelled OSM. Molecules which bind gratuitously, ie, without inducing the functional effects of OSM are most likely to be good antagonists. This assay may be reversed and labelled OSM receptor may be used with unlabelled OSM. A further screen with an ELISA format may be used to identify OSM antagonists where the ability of a candidate molecule to prevent binding of an OSM receptor conjugate such as gp130-Fc fusion protein to plate-immobilised OSM is measured, in this assay bound gp130-Fc is detected by enzyme-labelled anti-Fc antibody and colourimetric assay.
The functional effects of potential antagonists may be measured, for instance, by determining activity of a reporter system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of OSM or molecules that elicit the same effects on OSM. Reporter systems that may be useful in this regard include but are not limited to calorimetric labelled substrate converted into product, a reporter gene that is responsive to changes in the functional activity of OSM receptor, and binding assays known in the art.